Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of serine protease from neutrophils that is secreted by epithelial cells. We recently cloned mouse SLPI and discovered that this 12 kDa protein is also a product of neutrophils and macrophages and a component of human blood. SLPI can be induced locally as well as systemically in response to the microbial products lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from gram-negative and gram-positive bacteria, respectively. Expression of SLPI in mouse macrophages induces a hyporesponsive phenotype to both LPS and LTA. Moreover, we found that recombinant human SLPI suppresses the ability of human endothelial cells and neutrophils to respond to TNF by induction of adhesion molecules and secretion of reactive oxygen intermediates (ROI), respectively. Thus, SLPI has at least three anti-inflammatory functions: (i) inhibition of tissue-degrading neutrophil serine proteases; (ii) suppression of the production of inflammatory mediators by LPS- and LTA-stimulated mononuclear phagocytes; and (iii) suppression of inflammatory actions of TNF on endothelial cells and leukocytes. The goal of this project is to explore the mechanisms of the novel anti-inflammatory actions of SLPI: (ii) and (iii) above. The working hypothesis is that SLPI modulates inflammatory responses of macrophages, neutrophils and endothelial cells via a protease-independent mechanism, possibly mediated by its N-terminal domain. To test this hypothesis, we will focus on: (1) Characterization of the structural requirements for SLPI's novel anti-inflammatory effects by comparing the antiinflammatory activity of wild type SLPI, its anti-protease deficient mutants and its truncated forms (N-terminal or C-terminal half SLPI) using recombinant proteins and stably transfected cells expressing these proteins; (2) Identification of the molecular mechanisms involved in SLPI's anti-inflammatory functions by searching for specific molecules in Toll like receptor-signaling pathway affected by SLPI or specific protein targets associated with SLPI using surface crosslinking and yeast two hybrid screen; and (3) Testing the ability of exogenous and endogenous SLPI to protect mice in a endotoxic shock model using SLPI transgenic mice and administration of recombinant SLPI to septic mice. Elucidating how SLPI interferes with specific steps in LPS and LTA signaling may contribute important insights to the broader puzzle of the pathobiology of septic shock and in the development of novel strategies in its therapy.